Johnson W S, He Q Y, Tomasz M
Department of Chemistry, Hunter College, City University of New York, New York 10021, USA.
Bioorg Med Chem. 1995 Jun;3(6):851-60. doi: 10.1016/0968-0896(95)00067-q.
The clinically used natural antitumor agent mitomycin C (MC) is known to alkylate DNA monofunctionally and bifunctionally, resulting in the cross-linking of DNA. These reactions occur selectively with guanines at the CpG sequence. We show, confirming a previous report (Millard, J. T.; Beachy, T. M. Biochemistry 1993, 32, 12850) that cross-linking in oligonucleotides is further enhanced when the cytosines in CpG.CpG are 5-methylated to m5CpG.m5CpG. It is shown, furthermore, that guanines in m5CpG are monoalkylated two- to three-times faster than in CpG indicating that the m5C-induced rate enhancement occurs at the first, monoalkylation step of the two-step cross-linking process. The same MC-DNA adducts are formed in methylated as in non-methylated DNA. The basepaired but not the 5'-flanking, m5C residue is responsible for the enhanced alkylation of guanine. Enzymatically activated or Na2S2O4-activated MC shows identical rate-enhancement of alkylation at m5CpG. pBR322 DNA methylated by CpG-methylase was cross-linked two- to three-times more efficiently by MC than non-methylated DNA, indicating that the m5C effect is not an artifact of oligonucleotides. An electronic effect of the 5-methyl group of cytosine transmitted via G.C H-bonding to N2 of guanine is suggested as responsible for increased reactivity with MC. CpG is severely depleted in mammalian DNA and it is speculated that this factor attenuates MC cytotoxicity in human cells.
临床使用的天然抗肿瘤药物丝裂霉素C（MC）已知可单功能和双功能地使DNA烷基化，导致DNA交联。这些反应在CpG序列的鸟嘌呤处选择性发生。我们证实了之前的一份报告（Millard，J.T.；Beachy，T.M.《生物化学》1993年，32卷，12850页），即当CpG.CpG中的胞嘧啶被5-甲基化为m5CpG.m5CpG时，寡核苷酸中的交联会进一步增强。此外，研究表明，m5CpG中的鸟嘌呤比CpG中的鸟嘌呤单烷基化速度快两到三倍，这表明m5C诱导的速率增强发生在两步交联过程的第一步单烷基化步骤。甲基化和未甲基化的DNA中形成的MC-DNA加合物相同。碱基配对但不是5'-侧翼的m5C残基导致鸟嘌呤烷基化增强。酶激活或Na2S2O4激活的MC在m5CpG处显示出相同的烷基化速率增强。经CpG甲基化酶甲基化的pBR322 DNA被MC交联的效率比未甲基化的DNA高两到三倍，这表明m5C效应不是寡核苷酸的假象。推测胞嘧啶5-甲基基团通过G.C氢键传递给鸟嘌呤N2的电子效应是与MC反应性增加的原因。CpG在哺乳动物DNA中严重缺失，据推测，这一因素会减弱MC在人类细胞中的细胞毒性。
